The present invention relates to resonant sensors and in particular to biosensors useful for the qualitative and quantitative determination of one or more analytes, e.g., a specific protein, carbohydrate, glycoprotein, protein complex, nucleic acid molecules, (DNA and RNA) mycoplasma, virus, bacterium, yeast, mammalian cell, parasites, cysts, prions, or phospholipid, in a test sample suspected of containing such analyte(s).
A host of analytical techniques have been developed for the detection of biomolecules, pathogens and the like. Such techniques include surface plasmon resonance (SPR), polymerase chain reaction (PCR), enzyme-linked immunoassay (ELISA), and immuno-magnetic beads, to name a few. Although widely used, these techniques are not very sensitive and require extensive sample preparation, especially with complex or unpurified biological samples. Moreover, most biosensing platforms now in commercial use are not capable of making analyte determinations by measuring changes in mass on a sensor surface.
Considerable research has been devoted to the advancement of microscale biosensor technology, with a view toward achieving analyte detection with high specificity, sensitivity and reproducibility, but at a reduced cost compared to SPR, PCR, ELISA and the like.
A biosensor is a device in which a biological component, such as a nucleic acid probe, an antibody or other member of a specific binding pair, and a transducer or detector (i.e. optical, piezoelectric, electrochemical, etc.) component are integrated to generate a measurable signal. Biosensing devices have been proposed for use in a wide variety of applications, including medical diagnostics (determination/quantification of biomarkers present in bodily fluid), environmental testing (pathogens in drinking water), assessing food safety (Listeria, Cyrptosporidum, Giardia and E. coli contamination) and in biodefense (monitoring bioterrorism agents). One such device is a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor that measures a change in mass based on a resonance frequency shift, as described in detail in U.S. Patent Application Publication No. 2007/0169553 of Mutharasan et al. Briefly, the sensor consists of a piezoelectric (P) or active layer, e.g., lead zirconate titanate (PZT), adhesively bonded to a non-piezoelectric (NP) or passive layer of a few millimeters in length and 1 mm in width. The active layer is mounted in or on a suitable support by means of a mounting material, such as non-conducting epoxy. The active layer and the passive layer overlay, in shingle fashion, so that the respective layers are not coextensive. The PZT layer of the cantilever serves both as an actuating and as a sensing element. When an electric field is applied across the thickness of the PZT layer, it undergoes three dimensional deformation. Deformation occurs primarily along the planar dimensions of the PZT layer, because of geometrical and associated constraints, causing the base non-piezoelectric layer to flex. If the field alternates, the sensor experiences flexural oscillations. The sensor resonates when the excitation frequency coincides with the natural frequency (mechanical) of the cantilever beam. At resonance, the cantilever undergoes higher than normal bending stresses and the PZT layer, being electro-mechanically active, exhibits a sharp change in electrical impedance. The phase angle between the excitation voltage and the resulting current changes significantly, and is conveniently measured using an impedance analyzer. The sensing response is recorded by measuring changes in resonance frequency of the vibrating sensor.
PEMC sensors have been shown to be useful for detection of water-borne pathogens, such as E. coli, and for detection and direct quantification of protein-protein binding interactions. Campbell & Mutharasan, Biosensors and Bioelectronics, 21; 462-473 and 597-607 (2005). See also Campbell and Mutharasan, Anal. Chem., 79: 1145-52 (2007), which reports the measurement of B. anthracis in the presence of substantial concentrations of B. thuringiensis and B. cereus. 
PEMC sensors have a practical advantage over sensor platforms based on quartz crystal microbalance (QCM), involving a disk device that uses thickness-mode resonance for sensing. Although quartz is a weak piezoelectric material, it is widely used as a layer thickness monitor due in part to the availability of large quartz single crystals from which the membranes are made. The typical mass detection sensitivity of a 5 MHz QCM device having a minimum detectable mass density (DMD) of 10−9 g/cm2 is about 10−8 g/Hz, which is about four orders of magnitude less sensitive than PEMC devices. Thus, QCM analysis is of limited value when the analyte is present at low concentration together with a high level of contaminants.
It is also quite common to fabricate cantilever sensors, especially micro- and nano-scale cantilever sensors, using microelectromechanical systems (MEMS) or other wafer level etching techniques, whereby the cantilever “finger” is integral with, and of the same material as the base or anchor. Consequently, the stiffness of the “finger” and base will be identical so that resonant frequency modes, observed by changes in the “finger” impedance will be highly complicated and minimally useable. The effective mass of the sensor will also be relatively high, which will make the sensitivity comparatively low and any resonant frequencies will also be comparatively lower than sensors which are not integrally fabricated in this way.
Another piezoelectric cantilever-type sensor is described in U.S. Patent Application Publication No. 2007/0089515 of Shih et al. In an embodiment of that sensor intended for bio-detection, antibodies or other specific receptors of target antigens may be immobilized on the cantilever surface, preferably on the non-pizeoelectric tip. Binding of the target antigens to the cantilever surface increases the cantilever mass. Detection of target antigens is achieved by monitoring the cantilever's resonance frequency and determining the resonance frequency shift that is due to the mass of the adsorbed target antigens on the cantilever surface. The asymmetrical cantilever design, incorporating an overlapping non-peizoelectric tip, is described as enhancing the sensitivity of the sensor.
Although the piezoelectric cantilever sensors of the prior art are satisfactory in many respects, the excess length resulting from the overlap between the piezoelectric layer and non-piezoelectric layer adds parasitic geometry to the sensor which degrades key aspects of the sensor performance. The overlap portion in certain embodiments (P>NP), together with the mounting material and electrode attachment, increases the effective mass of the sensor, thus causing a corresponding reduction in sensitivity. Furthermore, the mounting material in the piezoelectric cantilever sensors of the prior art produces a damping effect, which decreases the quality (Q) factor of the signal produced, i.e. the ratio of resonance peak frequency to the resonance peak width at half peak height. The prior art sensors also vary in sensitivity along their length, which adds uncertainty to the measurement fidelity. The asymmetry of the prior art sensors also tends to introduce unwanted or degenerate modes into the detection signal. Damping is added to the prior art sensors, to convolute the degenerate modes. This can lead to variations in sensitivity, depending on the original separation of the modes and the location of the analyte receptors. Such variations can completely obscure the sensing signal or at least diminish the signal-to-noise ratio. Finally, the prior art sensors do not suggest where the vibrational nodes, present in all vibrating structures, are to be found. These nodes will be sites where the attachment of a target analyte will not register a sensor response. Hence, if these vibrational node sites are unknown, or are not excluded as a sensing region, the sensor will be less accurate and have greater variability, as compared with a sensor in which the node sites are identified and excluded from the sensing region.
Thus, a need exists for improved resonant sensor designs which provide sensitivity at least comparable to the biosensors of the prior art, with increased Q factor, and which can be fabricated with high reproducibility and at relatively low cost.